The leading cause of blindness in adults between the ages of 20 and 74 years is diabetic retinopathy (DR). Seven million people in the United States have diabetes. Diabetic retinopathy will affect the vast majority during their lifetime, with 8,000 to 40,000 of these people becoming blind each year. While management of diabetic retinopathy has improved as a result of landmark clinical trials, risk of complications, such as loss of visual acuity, loss of night vision and loss of peripheral vision, remains significant and treatment sometimes fails. Currently, laser photocoagulation is the most effective form of therapy for advanced disease.
Diabetic retinopathy is characterized by aberrant neovascularization of the retinal vasculature with edema and breakdown in the blood-retinal barrier (BRB) that leads to hemorrhage, tissue damage and retinal scarring. Unfortunately, current treatment options are inadequate and the disease is often progressive even with successful glucose control. An increasing body of evidence indicates that growth factor inhibitors offer the potential to treat a probable cause of diabetic retinopathy by blocking key mediating steps in disease progression.
A. Diabetic Retinopathy and Growth Hormone Inhibitors
Diabetic retinopathy is recognized as a retinal vascular disorder that includes: (i) excess capillary permeability, (ii) vascular closure, and (iii) proliferation of new vessels. The disease is characterized by a loss of retinal capillary pericytes, thickening of the basemerit membrane, microaneurysms, dot-blot hemorrhages, and hard exudates. The more severe form of the disease is proliferative retinopathy with extensive neovascularization, vessel intrusion into the vitreous, bleeding and scarring around new vessels that leads to severe vision impairment. However, the mechanisms of disease progression remain incompletely understood.
A half century ago, Michaelson postulated that humoral factors stimulated neovascularization in response to anoxia in his studies of retinal disease and an increasing body of evidence indicates growth factors play a pivotal role in progression of diabetic retinopathy.
Evidence linking increased growth hormone (GH) and diabetic retinopathy is substantial. An important role for growth hormone in diabetic retinopathy was indicated 40 years ago when Poulsen described DR regression in a post-partum woman with spontaneous pituitary infarction and proposed hypophysectomy to treat the disease. Controlled clinical trials have shown pituitary ablation could improve diabetic retinopathy and therapeutic success was correlated with the magnitude of growth hormone decrease. Additional support for the growth hormone hypothesis includes: (i) the observation that retinopathy accelerates during puberty when tissue sensitivity to GH is increased; (ii) diabetic patients with hemochromatosis and infiltrative destruction of the pituitary have little eye disease; and (iii) GH-deficient dwarfs with diabetes have no evidence of either macro or microvascular disease. Even diabetics with adequate glucose control show excess GH profiles and diabetic retinopathy is correlated with the magnitude of growth hormone hypersecretion.
Recognition that insulin-like growth factor 1 (IGF-1) mediates most of the anabolic effects of growth hormone has implicated IGF-1 in diabetic vascular complications. Several clinical studies support a role for IGF-1 in development of retinal neovascularization. Merimee and colleagues found increased serum IGF-1 levels from patients with rapidly accelerating diabetic retinopathy. A subsequent prospective study showed that patients had elevated IGF-1 serum levels at the time new retinal vessels first appeared compared to their serum IGF-1 levels three months before the onset of retinal neovascularization. In a large population-based study of 928 diabetic patients, higher levels of IGF-1 were correlated with an increased frequency of proliferative retinopathy.
However, other studies have shown that circulating IGF-1 levels are inappropriately low in most insulin dependent diabetes mellitus (IDDM) patients given their higher-than-normal growth hormone levels. The major source of circulating IGF-1 is the liver, where GH in the presence of insulin triggers IGF-1 gene transcription. IDDM patients have a lower IGF-1 response to exogenous GH, indicating a form of growth hormone resistance. An explanation for clinical studies that showed low IGF-1 patterns in IDDM but high IGF-1 in severe diabetic retinopathy may involve portal insulin levels in IDDM patients. Sönksen et al. recently suggested that the lower portal insulin in IDDM subjects (compared to levels seen by the liver during pancreatic insulin secretion) is responsible for decreased circulating IGF-1 in response to GH stimulation. Thus, endocrine conditions in insulin dependent diabetes mellitus are ideal for excess IGF-1 formation in local tissues, since high circulating levels of GH and insulin (an obligatory consequence of insulin injections in IDDM patients and insulin resistance in NIDDM patients) are available to stimulate IGF-1 local production in peripheral tissues, including the retina. The dynamics between endocrine serum IGF-1 and paracrine tissue IGF-1 production in IDDM subjects is not understood.
Yet other recent studies by Grant support paracrine/autocrine regulation of IGF-1 in diabetic retinopathy. Vitreous levels of IGF-1 better reflect the local levels of growth factors seen in retinal tissue and were measured in 23 diabetic patients with proliferative diabetic retinopathy and compared with age-matched control values. A 3-fold increase was observed in the DR samples compared with controls. IGF-1 secretion was augmented by basic fibroblast growth factor (b-FGF) in cultured human retinal endothelial cells, supporting a paracrine role. Other investigators have shown that IGF-1 receptors increase in retina from diabetic rats that are a model for IDDM.
IGF-1 appears to mediate retinal neovascularization. New vessel formation starts with basement membrane degradation followed by endothelial cell migration and proliferation. IGF-1 stimulates the release of tissue-type plasminogen activator (t-PA) from retinal endothelial cells derived from diabetic patients, but not from retinal endothelial cells derived from nondiabetic individuals. t-PA converts plasminogen to plasmin which can lyse thrombus as well as degrade most of the components of the extracellular matrix. Diabetic endothelial cells have a different response to growth factors. IGF-1 increases the expression of mRNA and protein for type IV collagenase in these same cells and acts synergistically with b-FGF on expression of both t-PA and type IV collagenase that are required for basement membrane degradation. IGF-1 receptors are present on retinal microvascular cells and these cells respond to IGF-1 with a five-fold increase in DNA synthesis. IGF-1 significantly promotes chemotaxis (migration) of human and bovine retinal endothelial cells and fetal bovine aorta endothelial cells in a dose-dependent manner. Thus, IGF-1 seems to act in concert with other growth factors in diabetic retinopathy.
Several studies indicate a role for b-FGF, endothelial growth factor (EGF) and transforming growth factor-α (TGF-α) in neovascularization. The precise functions of each of these factors in angiogenesis is yet to be elucidated. A common link may be response of the receptors upon binding these growth factors. IGF-1, insulin, b-FGF, platelet derived growth factor (PDGF), and EGF receptors belong to an expanded family of growth factor receptors, each sharing the common feature of a tyrosine kinase domain in the cytoplasmic portion of the molecule. Receptor binding induces autophosphorylation of the β-subunit of the receptor that activates the protein tyrosine kinase (PTK). Autophosphorylation renders PTK constituitively active, even when the growth factor is subsequently removed from the binding site. Consequently, dephosphorylation, and not merely dissociation of the growth factor, is required to terminate PTK activity. Activated PTKs are inactivated by protein tyrosine phosphatases (PTPases) which dephosphorylate the receptor.
The physiological inhibitor of GH and IGF-1 is somatostatin, a tetradecapeptide, found throughout the body, which has the structural formula:
Native somatostatin, however, has limited therapeutic use due to its extremely short half-life and wide range of inhibitory activities. Enzymatically stable somatostatin analogs have been developed with longer half-lives, such as octreotide, which is also known as sandostatin, and which has the structural formula: [SEQ ID NO.: 2]
and lanreotide (LNT), which has the structural formula: [SEQ ID NO.: 3]
In vitro studies have shown that the somatostatin analogs activate PTPases and therefore function at the biochemical level by promoting inactivation of the autophosphorylated growth factor receptor. Somatostatin and the somatostatin analogs do not cross the blood-brain barrier (BBB), which is consistent with their size, charge and lipid solubility.
Several somatostatin analogs are used for treating neuroendocrine neoplasms and as labeled diagnostic agents to identify tumor tissue. Preclinical studies suggest the agents may be therapeutically useful for treating restenosis following cardiac intervention procedures and chronic graft rejection which may be mediated in part by IGF-1 effects on smooth muscle cells. Clinical tolerance studies show that lanreotide is better tolerated than octreotide and the peptides appear to be equally effective in most preclinical and clinical studies.
The GH inhibitory and antiproliferative effects of somatostatin analogs stimulated several clinical trials in severe proliferative diabetic retinopathy. Initial clinical results were generally disappointing, but the studies were limited to a short dosing duration and doses that were inadequate to suppress GH or IGF-1 levels. More promising results were reported with lanreotide administered via continuous infusion pump for 4 weeks. The study enrolled 17 diabetic retinopathy patients with “low risk” proliferative disease and 8 of 11 subjects completed the lanreotide dosing schedule. Disease progression halted in 8 of 8 patients during lanreotide treatment (and disease regressed in 2 patients), while disease progressed in half the 6 control subjects. Retinal function improved during several months of continuous infusion with octreotide in an uncontrolled clinical trial with 4 patients.
Several factors suggest to the present inventors that drug efficacy could be improved by enhanced delivery of these peptides to the retina: (i) somatostatin receptors are present in retinal tissue that bind octreotide; (ii) octreotide and lanreotide do not cross the BBB; (iii) the inner retinal layer is protected by the BRB, the blood-retinal barrier; and (iv) the BRB is compromised in severe proliferative diabetic retinopathy where somatostatin analogs have shown efficacy.
One of the present inventors (Grant) has used octreotide in six patients (1987–1994) with severe proliferative diabetic retinopathy who previously received maximal pan-retinal photocoagulation, but still had persistent areas of neovascularization of the disk (NVD) and elsewhere (NVE). All six patients received octreotide in doses ranging from 25 to 100 μg three times a day. None of the six patients showed disease progression during their treatment period with octreotide and four patients had regression of their NVD. Whether this represented regression due to octreotide, a delayed response to pan-retinal photocoagulation, or spontaneous regression, is not known. Two of these patients have now been followed for more than 5 years. They continue to do well on octreotide.
A seventh diabetic retinopathy patient had severe bilateral neovascular glaucoma requiring treatment with bilateral Molteno implants in addition to retinal neovascularization. This patient's iris neovascularization regressed completely with the initiation of octreotide, but the posterior pole neovascularization was not affected. The drug was stopped because of gastrointestinal side effects and the iris vessels reappeared. The drug was restarted and there was complete regression of the iris neovascularization. Because the iris has no barrier which is equivalent to the BRB, these results support the hypothesis that somatostatin analogs can be effective in halting neovascularization when they reach their target site.
All seven study patients enjoyed an improved sense of well-being, reductions in their HgA1c, and improved visual acuity while on treatment with octreotide.
These encouraging clinical results led to a pilot study by Grant, begun in 1991, to establish whether octreotide can prevent the progression of diabetic retinopathy from either non-proliferative diabetic retinopathy or “low risk” proliferative diabetic retinopathy to “high risk” proliferative diabetic retinopathy. Previous studies have demonstrated that continuous infusion of somatostatin analogs has resulted in greater suppression of GH than intermittent injections. The current clinical study was designed to extend the study duration previously examined by McCombie and coworkers (one year rather than 8 weeks and continuous octreotide infusion with doses up to 5000 μg/day). The results to date are summarized below.
Of the four patients receiving octreotide, no patient showed clear clinical or angiographic improvement. Laser therapy was initiated in all four patients (in at least one eye) when diabetic retinopathy progressed to high risk characteristics as defined by the Diabetic Retinopathy Study recommendations. Suppression of GH and IGF-1 levels into the hypopituitary range was not achieved in any patient. While this inability to induce a “medical hypophysectomy” probably indicates that insufficient octreotide doses were used, a more important factor is whether adequate drug was delivered to retinal tissue.
In the control group (n=5), all patients showed disease progression to the point of requiring laser therapy in both eyes. Results suggest some positive somatostatin analog effects occurred; however, differences between the two groups in measured outcomes are not statistically significant. All four octreotide-treated subjects have shown normalized HgA1c within the first 6 months of therapy, which could not be achieved in the control group. In addition, the patients receiving octreotide had a statistically significant improvement in visual acuity that could be attributable to an improved blood glucose control.
Studies conducted on rapidly proliferating human retinal endothelial cells (HREC) that were stimulated with IGF-I and b-FGF in vitro have demonstrated direct inhibitory effects of somatostatin analogs in these cells. Octreotide results in a dose-dependent inhibition in [3H] thymidine incorporation measured in both IGF-1 and b-FGF stimulated HREC. This effect of octreotide was examined using HREC derived from diabetic and nondiabetic donors. Growth factors (IGF-1 and b-FGF) were found to stimulate HREC proliferation. Octreotide had minimal effect on cell proliferation in cells grown in control medium. Octreotide significantly decreased the proliferation of HREC in the IGF-1 stimulated cultures and this inhibitory effect was doubled to 40–45% in the b-FGF stimulated cultures. In summary, these in vitro studies support the presence of somatostatin receptors in human retinal tissue and demonstrate a direct inhibition by octreotide on both cell proliferation and DNA synthesis.
In view of the foregoing, it is apparent that a serious need exists for a means of delivering therapeutic concentrations of peptides having growth factor inhibitory activity to the retina in a site-specific and sustained manner for the prevention and treatment of diabetic retinopathy.
B. Blood Retinal Barrier (BRB) and Blood Brain Barrier (BBB) Functions
Several similarities exist between the BRB and the BBB. Zona occludens or tight junctions are the predominant structural feature of non-fenestrated endothelial cells in capillary beds from both brain and retina areas that exclude dye markers injected in the systemic circulation. In contrast, fenestrated capillary endothelium are found in brain and eye areas that permit dye penetration. Pericytes also contribute to barrier function for both the BRB and BBB. However, the simple lipid bilayer model is often used to explain barrier function since this model is consistent with the correlation between the octanol/water partition coefficients and brain uptake measured for many drugs.
The barrier function is not absolute since compounds do enter parenchymal tissue. Transcytosis occurs in both brain and retinal endothelial cells via carrier transport. Paracellular permeation and vesicular transport appear to play little if any role in barrier penetration. These similarities have been used to design an in vivo model to test carrier-mediated brain delivery of conjugated nerve growth factor with septal tissue transplants into the anterior eye chamber.
There is also evidence for functional differences between the BRB and BBB. First, there is clear evidence that the BRB is compromised in diabetes, but no evidence that the BBB is comparably damaged. Second, the few available transport and uptake studies of the BRB analogous to those developed to study the BBB suggest differences. Two research groups have shown a higher permeability surface area product for sucrose in the retina than the brain while another group found no difference. A recent study in normal rats concluded that the BRB is approximately 4 times as permeable to a labeled amino acid compared to the BBB. The investigators concluded that the more numerous pericytes may play an increased protective function for the retina. Pericytes decrease relatively early in diabetes. Third, systemically administered drugs are more effective in reaching retinal tissue than the CNS. An example is the case report for an AIDS patient treated for cytomegalovirus (CMV) retinitis and encephalitis with ganciclovir. Despite successful treatment of CMV retinitis verified at autopsy, there was widespread CMV-encephalitis. Finally, some histocytochemical differences in the vascular beds indicate functional differences between the BBB and BRB, as shown in the following table:
GENERAL FEATURE COMPARISON OF MICROVESSELSIN RAT EVE AND BRAINBrainRetinaIrisCiliary bodyLuminal diameter (μm) 4.4 ± 0.11 4.8 ± 0.23 6.7 ± 0.28 7.4 ± 0 24Wall thickness (μm)0.33 ± 0.020.39 ± 0.020.46 ± 0.020.45 ± 0.02Cytoplasmic area/profile (μm2) 4.2 ± 0.34 7.0 ± 0.8711.4 ± 0.6511.1 ± 0.08Fenestrations (#/100 μm perimeter)00031.6 ± 3.92Junctions with wide clefts (%)0011.818Pericyte area/profile (μm2) 1.4 ± 0.21 3.8 ± 0.54not measurednot measuredMean ± SEM data after Stewart, P.A. and Tuor, U.I. (1994) J. Comp. Neurol. 340, 566–576.
All of these functional differences suggest that drug delivery could be designed to preferentially enhance BRB penetration without significant BBB interaction.
C. Redox-Based Chemical Delivery System (CDS) for Brain Targeting of Centrally Acting Drugs
Various strategies have been developed for directing centrally acting drugs, including in some cases peptides, into the brain. A dihydropyridine⇄pyridinium redox system has recently been successfully applied to delivery to the brain of a number of drugs. Generally speaking, according to this system, a dihydropyridine derivative of a biologically active compound is synthesized, which derivative can enter the CNS through the blood-brain barrier following its systemic administration. Subsequent oxidation of the dihydropyridine species to the corresponding pyridinium salt leads to delivery of the drug to the brain.
Five main approaches have been used thus far for delivering drugs to the brain using a redox system. The first approach involves derivation of selected drugs which contain a pyridinium nucleus as an integral structural component. This approach was first applied to delivering to the brain N-methylpyridinium2-carbaldoxime chloride (2-PAM), the active nucleus of which constitutes a quaternary pyridinium salt, by way of the dihydropyridine latentiated prodrug form thereof. Thus, a hydrophilic compound (2-PAM) was made lipoidal (i.e. lipophilic) by making its dihydropyridine form (Pro-2-PAM) to enable its penetration through lipoidal barriers. This simple prodrug approach allowed the compound to get into the brain as well as other organs, but this manipulation did not and could not result in any brain specificity. On the contrary, such approach was delimited to relatively small molecule quaternary pyridinium ring-containing drug species and did not provide the overall ideal result of brain-specific, sustained release of the desired drug, with concomitant rapid elimination from the general circulation, enhanced drug efficacy and decreased toxicity. No “trapping” in the brain of the 2-PAM formed in situ resulted, and obviously no brain-specific, sustained delivery occurred as any consequence thereof: the 2-PAM was eliminated as fast from the brain as it was from the general circulation and other organs. Compare U.S. Pat. Nos. 3,929,813 and 3,962,447; Bodor et al, J. Pharm. Sci., 67, No. 5, 685 (1978). See also Bodor, “Novel Approaches for the Design of Membrane Transport Properties of Drugs”, in Design of Biopharmaceutical Properties Through Prodrugs and Analogs, Roche, E. B. (ed.), APhA Academy of Pharmaceutical Sciences, Washington, D.C., 98–135 (1976). Subsequent extension of this first approach to delivering a much larger quaternary salt, berberine, to the brain via its dihydropyridine prodrug form was, however, found to provide site-specific sustained delivery to the brain of that anticancer agent. See Bodor et al, Science, Vol. 214, Dec. 18, 1981, pp. 1370–1372. This approach is not applicable to the delivery of peptides, however, since they do not comprise active quaternary pyridinium salts.
The second approach for delivering drugs to the brain using a redox system involves the use of a dihydropyridine/pyridinium carrier chemically linked to a biologically active compound. Bodor et al, Science, Vol. 214, Dec. 18, 1981, pp. 1370–1372, outlines a scheme for this specific and sustained delivery of drug species to the brain, as depicted in the following
According to the scheme in Science, a drug [D] is coupled to a quaternary carrier [QC]+ and the [D-QC]+ which results is then reduced chemically to the lipoidal dihydro form [D-DHC]. After administration of [D-DHC] in vivo, it is rapidly distributed throughout the body, including the brain. The dihydro form [D-DHC] is then in situ oxidized (rate constant, k1) (by the NAD⇄NADH system) to the ideally inactive original [D-QC]+ quaternary salt which, because of its ionic, hydrophilic character, should be rapidly eliminated from the general circulation of the body, while the blood-brain barrier should prevent its elimination from the brain (K3>>k2; k3>>k7). Enzymatic cleavage of the [D-QC]+ that is “locked” in the brain effects a sustained delivery of the drug species [D], followed by its normal elimination (k5), metabolism. A properly selected carrier [QC]+ will also be rapidly eliminated from the brain (k6>>k2). Because of the facile elimination of [D-QC]+ from the general circulation, only minor amounts of drug are released in the body (k3>>k4); [D] will be released primarily in the brain (k4>k2). The overall result ideally will be a brain-specific sustained release of the target drug species. Specifically, Bodor et al worked with phenylethylamine as the drug model. That compound was coupled to nicotinic acid, then quaternized to give compounds of the formula
which were subsequently reduced by sodium dithionite to the corresponding compounds of the formula
Testing of the N-methyl derivative in vivo supported the criteria set forth in Scheme A. Bodor et al speculated that various types of drugs might possibly be delivered using the depicted or analogous carrier systems and indicated that use of N-methylnicotinic acid esters and amides and their pyridine ring-substituted derivatives was being studied for delivery of amino- or hydroxyl-containing drugs, including small peptides, to the brain. No other possible specific carriers were disclosed. Other reports of this work with the redox carrier system have appeared in The Friday Evening Post, Aug. 14, 1981, Health Center Communications, University of Florida, Gainesville, Fla.; Chemical & Engineering News, Dec. 21, 1981, pp. 24–25; and Science News, Jan. 2, 1982, Vol. 121, No. 1, page 7. Subsequently, the redox carrier system was substantially extended in terms of possible carriers and drugs to be delivered. See International Patent Application No. PCT/US83/00725, filed May 12, 1983 and published Nov. 24, 1983 under International Publication No. W083/03968. Also see Bodor et al, Pharmacology and Therapeutics, Vol. 19, No. 3, pp. 337–386 (1983); and Bodor U.S. Pat. No. 4,540,564, issued Sep. 10, 1985.
The aforementioned Bodor U.S. Pat. No. 4,540,564 specifically contemplates application of the dihydropyridine⇄pyridinium salt carrier system to amino acids and peptides, particularly small peptides having 2 to 20 amino acid units. Among the amino acids and peptides mentioned in the patent are GABA, tyrosine, tryptophan, met5-enkephalin, leu5-enkephalin, LHRH and its analogs and others. Thus, in the carrier system as applied to amino acids and peptides, the free carboxyl function is protected in an effort to prevent premature metabolism, e.g. with an ethyl ester, while the trigonelline-type carrier is linked to the amino acid or peptide through its free amino function. Oxidation of the dihydropyridine carrier moiety in vivo to the ionic pyridinium salt carrier/drug entity prevents elimination thereof from the brain, while elimination from the general circulation is accelerated, and subsequent cleavage of the quaternary carrier/drug species results in sustained delivery of the amino acid or peptide (e.g. tryptophan, GABA, leu5-enkephalin, etc.) in the brain and facile elimination of the carrier moiety. This method is quite useful for delivery of amino acids; in the case of peptides, however, the typical suggested carboxyl protecting groups do not confer sufficient lipophilicity on the peptide molecule. Moreover, this approach does not address the problem of the enzymatic blood-brain barrier or suggest a means of avoiding that problem.
The third approach for delivering drugs to the brain using a redox system provides derivatives of centrally acting amines in which a primary, secondary or tertiary amine function has been replaced with a dihydropyridine/pyridinium salt redox system. These brain-specific analogs of centrally acting amines have been described in International Patent Application No. PCT/US85/00236, filed Feb. 15, 1985 and published Sep. 12, 1985 under International Publication No. W085/03937. The dihydropyridine analogs are characterized by the structural formula
wherein D is the residue of a centrally acting primary, secondary or tertiary amine, and
is a radical of the formula
These dihydropyridine analogs act as a delivery system for the corresponding biologically active quaternary compounds in vivo. Due to its lipophilic nature, the dihydropyridine analog will distribute throughout the body and has easy access to the brain through the blood-brain barrier. Oxidation in vivo will then provide the quaternary form, which will be “locked” preferentially in the brain. In contradistinction to the drug-carrier entities described in Bodor U.S. Pat. No. 4,540,564 and related publications, however, there is no readily metabolically cleavable bond between drug and quaternary portions, and the active species delivered is not the original drug from which the dihydro analog was derived, but rather is the quaternary analog itself.
The aforementioned International Publication No. WO85/03937 contemplates application of its analog system to amino acids and small peptides, e.g., the enkephalins, tryptophan, GABA, LHRH analogs and others. In this analog system as applied to amino acids and peptides, the free carboxyl function is thus protected to prevent premature metabolism while the dihydropyridine⇄pyridinium salt type redox system replaces the free amino function in the amino acid or peptide.
As described in International Publication No. WO85/03937, the chemical processes for preparing the redox analog derivatives replace any free amino function in the selected drug with the redox analog system. When these processes are applied to amino acids, they provide a redox amino acid which no longer contains a free amino function for linkage to another amino acid or peptide via a peptide bond (—CONH—). Such an analog amino acid can thus only be used to prepare a peptide having the analog amino acid located at the peptide's N-terminus. This limits use of the redox analog amino acids in peptide synthesis. Moreover, as noted hereinabove, this approach is not designed to ultimately deliver the original peptide to the brain, since there is no cleavable bond between peptide and quaternary portions; rather, the redox portion in this approach becomes an inherent, essentially inseparable part of a new peptide analog. Furthermore, this approach does not address the problem of the enzymatic blood-brain barrier or suggest a means for avoiding the premature degradation caused by the highly active neuropeptide degrading enzymes.
The fourth redox approach is designed to provide redox amino acids which can be used to synthesize peptides having a redox analog system inserted at a variety of locations in the peptide chain, including non-terminal positions, and has been described in Bodor U.S. Pat. No. 4,888,427, issued Dec. 19, 1989. These amino acids contain a redox system appended directly or via an alkylene bridge to the carbon atom adjacent to the carboxyl carbon. The peptides provided by U.S. Pat. No. 4,888,427 have an amino acid fragment of the formula
incorporated therein at a non-critical position in the peptide chain, i.e., at a position which is not critical to the pharmacological effect of the peptide. The final redox peptide of U.S. Pat. No. 4,888,427 preferably contains a total of 2 to 20 amino acid units. Typically, except for the presence of at least one redox amino acid fragment of structure (A) or (B) and the possible protection of terminal amino and carboxyl functions, the structure of the redox peptide is identical to that of a known, naturally occurring bioactive peptide or of a known bioactive synthetic peptide (particularly one which is an analog of a naturally occurring bioactive peptide).
It is apparent from the foregoing, that the fourth redox approach, like the third approach discussed above, is not designed to ultimately deliver the original peptide to the brain because there is again no cleavable bond between peptide and quaternary portions. Again, the redox system becomes an integral part of a new peptide analog, not a means for ultimately delivering the original peptide to the brain. Still further, this approach also does not address the problem of the enzymatic blood-brain barrier or suggest a means for avoiding deactivation of the peptide by enzymes before it achieves its therapeutic objective.
The fifth and most recent redox approach provides for brain-enhanced delivery of neuroactive peptides by sequential metabolism. This approach provides a means for “molecular packaging” of peptides which addresses the problems of the physical blood-brain barrier as well as the problems of the enzymatic blood-brain barrier. According to this fifth redox approach, a pharmacologically active peptide is placed in a molecular environment which disguises its peptide nature. This environment provides a biolabile, lipophilic function to penetrate the blood-brain barrier by passive transport; a dihydropyridine-type redox moiety for targeting the peptide to the brain and providing “lock-in” as the pyridinium salt; and an amino acid or di- or tripeptide spacer between redox moiety and peptide designed to enhance the sequential metabolism of the “molecularly packaged” peptide.
Consistent with the foregoing, the fifth redox approach provides, for brain-targeted peptide delivery, “packaged” peptide systems of the formula
wherein Z is either a direct bond or C1–C6 alkylene and can be attached to the heterocyclic ring via a ring carbon atom or via the ring nitrogen atom; R1 is C1–C7 alkyl, C1–C7 haloalkyl or C7–C12 aralkyl when Z is attached to a ring carbon atom; R1 is a direct bond when Z is attached to the ring nitrogen atom; R2 and R3, which can be the same or different, are selected from the group consisting of hydrogen, halo, cyano, C1–C7 alkyl, C1–C7 alkoxy, C2–C8 alkoxycarbonyl, C2–C8 alkanoyloxy, C1–C7 haloalkyl, C1–C7 alkylthio, C1–C7 alkysulfinyl, C1–C7 alkylsulfonyl, —CH═NOR′″ wherein R′″ is hydrogen or C1–C7 alkyl, and CONR′R″ wherein R′ and R″, which can be the same or different, are each hydrogen or C1–C7 alkyl; or one of R2 and R3 together with the adjacent ring carbon atom forms a benzene ring fused to the heterocyclic ring, which benzene ring may optionally bear one or two substituents, which can be the same or different, selected from the group consisting of hydroxy, protected hydroxy, halo, cyano, C1–C7 alkyl, C1–C7 alkoxy, C2–C8 alkoxycarbonyl, C2–C8 alkanoyloxy, C1–C7 haloalkyl, C1–C7 alkylthio, C1–C7 alkylsulfinyl, C1–C7 alkylsulfonyl, —CH═NOR′″ wherein R′″ is hydrogen or C1–C7 alkyl, and —CONR′R″ wherein R′ and R″, which can be the same or different, are each hydrogen or C1–C7 alkyl; the dotted lines indicate that the compound of formula (I) contains a 1,4- or 1,6-dihydropyridine, a 1,4- or 1,2-dihydroquinoline, or a 1,2-dihydroisoquinoline ring system; “spacer” is an L-amino acid unit or a di- or tripeptide consisting of 2 or 3 L-amino acid units, the N-terminal amino acid of said spacer being bonded to the depicted carbonyl carbon via an amide bond; and “peptide” is a pharmacologically active peptide having 2 to 20 amino acid units, the N-terminal amino acid of said peptide being bonded to the C-terminal amino acid of said spacer via a peptide bond, the C-terminal amino acid of said peptide having an esterified carboxyl function —COOR4 wherein R4 is C8–C22 alkyl, C8–C22 alkenyl, C6–C30 polycycloalkyl-CpH2p— wherein p is 0, 1, 2 or 3, or C6–C30 polycycloalkenyl-CpH2p— wherein p is defined as above. See, for example, Bodor U.S. Pat. No. 5,624,894, issued Apr. 29, 1997, incorporated by reference herein in its entirety and relied upon.
In specific embodiments of the brain-targeted molecular packaging of the aforementioned U.S. Pat. No. 5,624,894, packaged TRH-type peptides and enkephalin-type peptides are described and shown to deliver pharmacologically significant amounts of the peptides to the brain. As is apparent from the foregoing, both the COOH-terminus and the NH2-terminus of the peptide molecule are modified in such a way as to increase the lipid solubility of the peptide, and also to prevent cleavage by the BBB aminopeptidases. Additionally, the representative 1,4-dihydrotrigonellinate redox targetor (T) exploits the unique architecture of the BBB which allows for the influx of the lipid soluble neutral form, but is not permeable to the positively charged form. The enkephalins are sensitive to cleavage by endopeptidases at the Gly3-Phe4 peptide bond. Cholesteryl, a bulky and lipophilic steroidal moiety (L), provides a representative ester function that increases the lipid solubility and also hinders the C-terminal portion of the peptide from being recognized by peptide-degrading enzymes. This part of the molecule is, however, labile toward esterase or lipase, which permits its removal after delivery. The lipases or esterases expose the peptide unit that can interact with specific receptors, or that may serve as a substrate for various neuropeptide processing and degrading enzymes. A spacer function (S) is also incorporated in order to preserve the integrity of the peptide unit by spatially separating the important segment of the molecule (important in terms of central activity) from the targetor (T). In essence, the peptide unit of this brain delivery system appears as a pertubation on a bulky molecule dominated by the lipophilic steroidal portion and the targetor, which also prevents recognition by the peptidases.
The “packaged” peptide system of U.S. Pat. No. 5,624,894 thus offers a means of delivering neuropeptides to the brain. There is no suggestion therein, however, that the system could be adapted to the targeting of peptides to tissues of the body other than the brain.